Citrus Extract as a Perspective for the

Control of Dyslipidemia: A Systematic

Review With Meta-Analysis From

Animal Models to Human Studies

Betina M. R. Carvalho ¹, Laranda C. Nascimento ¹, Jessica C. Nascimento ¹,

Vitória S. dos S. Gonçalves ², Patricia K. Ziegelmann ³, Débora S. Tavares ⁴ and

Adriana G. Guimarães ⁵*

1Programa de Pós-Graduação em Ciências Aplicadas à Saúde, Universidade Federal de Sergipe, Lagarto, Brazil, 2Departamento

de Química, Universidade Federal de Sergipe, São Cristóvão, Brazil, ³Departamento de Estatística, Programa de Pós-graduação

em Epidemiologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, ⁴Departamento de Educação em Saúde,

Universidade Federal de Sergipe, Lagarto, Brazil, ⁵Departamento de Farmácia, Universidade Federal de Sergipe, São Cristóvão,

Brazil

This study aims to obtain scientific evidence on the use of Citrus to control dyslipidemia.

The surveys were carried out in 2020 and updated in March 2021, in the PubMed, Scopus,

LILACS, and SciELO databases, using the following descriptors: Citrus, dyslipidemias,

hypercholesterolemia, hyperlipidemias, lipoproteins, and cholesterol. The risk of bias was

assessed according to the Cochrane methodology for clinical trials and ARRIVE for

preclinical trials. A meta-analysis was performed using the application of R software. A

total of 958 articles were identified and 26 studies demonstrating the effectiveness of the

Citrus genus in controlling dyslipidemia were selected, of which 25 were included in the

meta-analysis. The effects of Citrus products on dyslipidemia appear consistently robust,

acting to reduce total cholesterol, LDL, and triglycerides, in addition to increasing HDL.

These effects are associated with the composition of the extracts, extremely rich in

antioxidant, as flavonoids, and that act on biochemical targets involved in lipogenesis and

beta-oxidation. The risk of bias over all of the included studies was considered critically low

to moderate. The meta-analysis demonstrated results favorable to control dyslipidemia by

Citrus products. On the other hand, high heterogeneity values were identified, weakening

the evidence presented. From this study, one can suggest that Citrus species extracts are

potential candidates for dyslipidemia control, but more studies are needed to increase the

strength of this occurrence.

Keywords: dyslipidemia, citrus, hyperlipidemia, flavonoids, cholesterol

Systematic Review Registration: [https://www.crd.york.ac.uk/prospero/display_record.php?ID=

CRD42019121238], identifier [PROSPERO 2019 CRD42019121238].

INTRODUCTION

Dyslipidemia has high rates of occurrence in the world population (Pirillo et al., 2021), being closely

related to obesity, metabolic syndrome (Mach et al., 2020), atherosclerosis (Wiggins et al., 2019),

Edited by:

Irwin Rose Alencar de Menezes,

Regional University of Cariri, Brazil

Reviewed by:

Amir Hadi,

Isfahan University of Medical

Sciences, Iran

Praveen Kumar M,

Nference, India

*Correspondence:

Adriana G. Guimarães

adrianagibara@hotmail.com

adrianagibara@pq.cnpq.br

Specialty section:

This article was submitted to

Gastrointestinal and Hepatic

Pharmacology,

a section of the journal

Frontiers in Pharmacology

Received: 26 November 2021

Accepted: 10 January 2022

Published: 14 February 2022

Citation:

Carvalho BMR, Nascimento LC,

Nascimento JC, Gonçalves VSS,

Ziegelmann PK, Tavares DS and

Guimarães AG (2022) Citrus Extract as

a Perspective for the Control of

Dyslipidemia: A Systematic Review

With Meta-Analysis From Animal

Models to Human Studies.

Front. Pharmacol. 13:822678.

doi: 10.3389/fphar.2022.822678

Frontiers in Pharmacology | www.frontiersin.org

February 2022 | Volume 13 | Article 822678

1

SYSTEMATIC REVIEW

published: 14 February 2022

doi: 10.3389/fphar.2022.822678

coronary heart disease (Zhao et al., 2021), increased susceptibility

to cancer (Khan et al., 2021), and more recently increased

mortality and severity of COVID-19 (Atmosudigdo et al.,

2021). This disorder is characterized by changes in the lipid

profile, including an increase in total serum cholesterol, low-

density lipoprotein (LDL-c), and triglycerides, as well as a

decrease in high-density lipoprotein (HDL-c) rates in the

blood (Fruchart et al., 2008). The relationships between these

markers have been used as indicators of insulin resistance and

metabolic disorders (Sowndarya et al., 2021), in addition to

atherosclerosis and coronary heart disease (Abid et al., 2021).

However, inflammation markers such as us-CRP (high serum

sensitivity C-reactive protein) can also be considered important

indicators to estimate the severity and risk of coronary artery

disease (Patil et al., 2020). Although there are therapeutic options

for the treatment of dyslipidemias, these are not fully effective,

due to non-adherence to treatment by various factors such as

adverse

effects,

intolerance,

regimen

complexity,

and

imperceptible benefits, besides the need to combine drugs to

improve the clinical condition (Schulz, 2006; Ingersgaard et al.,

2020). On the other hand, lipid-lowering drugs are still

inaccessible to the majority of the population in low-income

countries (Pirillo et al., 2021), making the search for new

strategies to control dyslipidemia necessary.

In this sense, searching for new treatment strategies for this

important health problem is necessary. In this perspective, several

plants and natural products have been studied regarding their

effects on dyslipidemia control (Ballard et al., 2019; Adel

Mehraban et al., 2021); among them, the species of the genus

Citrus (Lamiquiz-Moneo et al., 2020) stand out. Belonging to the

Rutaceae family, the genus Citrus is widely distributed in tropical

and subtropical regions (Manuel et al., 2020) and contains several

substances with biological and nutritional potential, such as fibers

(e.g., pectin), vitamins, and bioactive compounds, with emphasis

on the flavonoids (Alam et al., 2013; Rafiq et al., 2018). Naringin,

naringenin, nobiletin, narirutin, and hesperidin correspond to the

most frequently found flavonoids. They have pronounced

antioxidant and anti-inflammatory activities (Tripoli et al.,

2007; Craft et al., 2012), in addition to being effective in

controlling metabolic syndromes, lipid changes, and obesity

(Geleijnse et al., 1999; Lee et al., 2001; Gattuso et al., 2007;

Alam et al., 2013; Sahebkar, 2017; Ballard et al., 2019).

Thus, this review sought to compile the scientific findings that

demonstrate the effect of Citrus extracts on the control of serum

lipid levels, measuring the size of the effect through meta-analysis.

MATERIAL AND METHODS

Focused Question

The question to be answered was established from the

bibliographic survey “Are species of the genus Citrus effective

in reducing dyslipidemia?” conducted through four steps: (Pirillo

et al., 2021) identification of the use of the Citrus species, (Mach

et al., 2020) identification of the pathology to be applied

(dyslipidemia), (Wiggins et al., 2019) definition of the types of

studies included (preclinical and clinical), and (Zhao et al., 2021)

definition of the target outcome to be analyzed, which is the lipid

profile, building the PICOS strategy (patient or pathology,

intervention, control, other outcomes, and the type of study).

PICOS is highlighted as follows: P: dyslipidemia; I: species of the

genera Citrus (extract); C: untreated or placebo-treated and

hyperlipidemia-induced group; O: blood lipid levels; and S:

preclinical or clinical studies.

Review Writing and Registration of

Protocols

The writing of this systematic review was based on the

recommendations

of

the

Preferred

Reporting

Items

for

Systematic Reviews and Meta-Analyses (PRISMA) (Page et al.,

2021) tool. In addition, the instrument that guides how the

experimental studies should be analyzed was ARRIVE (Animal

Research:

Reporting

of

In

Vivo

Experiments)

guidelines

(Kilkenny et al., 2010). The protocol for this review was

registered

in

the

International

Prospective

Register

Systematic Reviews (Prospero) database and registered on the^of

website https://www.crd.york.ac.uk/prospero/, through approved

registry No. CRD42019121238.

Literature Search

The search was carried out through search strategies in the

LILACS, PubMed, SciELO, and Scopus databases in 2019 and

updated in March 2021. The terms used to compose the search in

the databases were defined from consultations with MeSH and

DeCS descriptors. Thus, the following search strategy was

structured: “CITRUS” AND “Lipoproteins” OR “Cholesterol”

OR

“Epicholesterol”

OR

“Dyslipidemias”

OR

“Dyslipoproteinemia” OR “Hypercholesterolemia” OR “High

Cholesterol Levels” OR “Hyperlipidemias” OR “Lipidemia,”

described in detail in Supplementary Table S1.

Study Selection and Eligibility Criteria

After excluding duplicate records, titles, abstracts, and full texts

were independently analyzed by two researchers in order to

determine the study’s eligibility for inclusion in the review.

The inclusion criteria were preclinical studies or randomized

clinical trials that include the use of Citrus species to assess the

effect on the lipid profile. In this review, were excluded reviews,

case studies, case reports, and studies that did not assess the

action on the lipid profile, which included the use of juices from

Citrus species and their action on the lipid profile, or the

association of Citrus species with another compound that

could modify the lipid profile, as well as studies that used

compounds

isolated

from

Citrus

species

to

target

hyperlipidemia. To assess the agreement among researchers,

the statistical test of the Kappa coefficient (K) was applied.

Data Extraction and Risk of Bias

Assessment

Two independent reviewers extracted data from the included

studies. The data from preclinical studies were as follows: Citrus

species, type of extract and part of the plant, composition,

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia

hyperlipidemia induction model, evaluation methods, treatment,

animal species, and results (all results that were in mg/dL were

converted to mmol/L using the OnlineConversion.com electronic

calculator according to the type of cholesterol). The data from

clinical studies were as follows: Citrus species, type of extract and

part of the plant, composition, study design/location, sample,

criteria for inclusion and exclusion of participants, pathologies,

treatment, and results (all results that were in mmol/L were

converted

to

mg/dL

using

the

OnlineConversion.com

electronic calculator according to the type of cholesterol). All

the outcomes of the experiments carried out in the articles were

extracted for descriptive and inferential analyses.

Through ARRIVE, we apply the following: precise and concise

description of the content of the article in the title, abstract,

explanation of the methodological approach of the introduction,

general and specific objectives, ethical nature of care and use of

animals, study design regarding the number of animals per group,

experimental procedures, information about animals such as sex,

size, weight, and age, housing and breeding, sample size,

statistical

methods,

description

of

results

and

their

interpretation, and study funding.

All clinical studies included in this research were approved for

methodological quality in the risk checklist of Cochrane

randomized for controlled trials (Cochrane Training, 2019).

Items such as generation of random sequence, concealment of

allocation, certification of participants and professionals, as well

as of evaluators, incomplete and selective outcomes, or whether

the study presents any other problem or fraud were used. The

studies considered as having the highest methodological quality

were those related to randomization, blinding, and complete

outcomes.

Meta-Analysis

The studies selected for the meta-analysis had the following

outcomes

analyzed:

total

cholesterol,

LDL,

HDL,

and

triglyceride levels, including the baseline and post-treatment

data from both the control and treatment groups for both

preclinical and clinical studies. In addition to the primary

outcomes,

to

improve

the

understanding

of

the

effects

observed in preclinical studies, the studies were separated into

the following subgroups: route of administration of the extract,

type of animal, type of extract, and parts of the plant used.

For the quantitative analysis of the articles, the studies selected

presented the value of the sample n, mean, deviation, or standard

error for the serum levels of total cholesterol, LDL, HDL, and/or

triglycerides of the treatment and control groups. All data were

tabulated in Excel and later analyzed using the application of R

software. The heterogeneity of the studies was measured using

Cochran’s Q test, using the I² statistic, which was considered as

heterogeneous when the p value was less than 0.05. The

heterogeneity between the studies was defined using the I²

statistic, which was considered with an unimportant (I² <

25%), moderate (25% < I² < 75%), or high degree of

heterogeneity (I² > 75%) (Higgins and Thompson, 2002). For

heterogeneous studies (I² > 75%), the following subgroup

analyses were performed: route of administration, type of

animal, parts of the plant used in the extract, type of fruit,

and type of extract. In addition, we performed a sensitivity

analysis,

sequentially

removing

the

individual

studies

to

determine whether any single study affected the overall effect

estimate.

RESULTS

Study Selection and Study Characteristics

During the search process, 958 articles were obtained: 169 from

PubMed, 762 from SciVerse Scopus, 12 from SciELO, and 15

from LILACS. After analyzing the titles, 598 duplicate articles

were removed. After excluding the repeated articles, 360 titles

were screened for analysis according to the inclusion criteria,

from which 329 studies were excluded for not inducing

hyperlipidemia

in

an

animal

model

or

for

not

having

dyslipidemia installed in the case of clinical studies. In

addition, studies with isolated compounds of the Citrus species

or without evaluation of total cholesterol, LDL-C, HDL-C, or

triglycerides were also excluded.

After this design, 31 articles remained, the full texts of which

were analyzed, thus yielding 27 articles that were finally included

in the qualitative synthesis (Figure 1; Tables 1–3). Of these, 22

studies were preclinical trials (Vinson et al., 1998; Bok et al., 1999;

Terpstra et al., 2002; Zulkhairi et al., 2010; Ding et al., 2012; Kang

et al., 2012; Raasmaja et al., 2013; Lu et al., 2013; Kim et al., 2013;

Muhtadi et al., 2015; Dinesh and Hegde, 2016; Shin et al., 2016;

Ashraf et al., 2017; Fayek et al., 2017; Chou et al., 2018; Feksa

et al., 2018; Mir et al., 2019; Sato et al., 2019; Hase-Tamaru et al.,

2019; Ling et al., 2020; Ke et al., 2020; Lee et al., 2020), 3 were

exclusively clinical studies (Gorinstein et al., 2007; Toth et al.,

2015; Cai et al., 2017) and 1 study contained preclinical and

clinical protocols (Mollace et al., 2011) (Figure 1). For the

quantitative synthesis, 25 articles (Vinson et al., 1998; Bok

et al., 1999; Gorinstein et al., 2007; Zulkhairi et al., 2010;

Mollace et al., 2011; Ding et al., 2012; Kang et al., 2012;

Terpstra et al., 2012; Kim et al., 2013; Lu et al., 2013;

Raasmaja et al., 2013; Muhtadi et al., 2015; Dinesh and Hegde,

2016; Shin et al., 2016; Ashraf et al., 2017; Cai et al., 2017; Fayek

et al., 2017; Chou et al., 2018; Feksa et al., 2018; Hase-Tamaru

et al., 2019; Mir et al., 2019; Sato et al., 2019; Ke et al., 2020; Lee

et al., 2020; Ling et al., 2020) were selected. The level of agreement

among the reviewers was 0.470, being considered as moderate.

Tables 2 and 3 show the general characteristics and results of

the preclinical studies, arranged in the chronological order of

publication. Table 4 present the experimental conditions and

results of clinical trials also arranged in the chronological order.

The selected articles were published between 1998 and 2020,

with a predominance of the number of publications in 2013 (n =

3), 2017 (n = 3), 2019 (n = 3), and 2020 (n = 3). These studies were

conducted mainly in China (n = 6; 23.0%) and Korea (n = 5;

19.2%) followed by Italy (n = 2; 7.6%) and Japan (n = 2; 7.6%), in

addition to other countries in which only 1 study was found as

described in Tables 1–3.

In the 26 selected articles, 15 different species of Citrus

were studied in a dyslipidemia model: C. reticulata (n = 4;

15.3%), C. bergamia (n = 3; 11.5%), C. sinensis (n = 3; 13.6%),

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia

C. junos Tanaka (n = 2; 9.1%), C. grandis (L.) Osbeck also

called C. maxima (n = 3; 11.5%), C. paradise also known as

grapefruit (n = 2; 7.6%), C. unshiu (n = 2; 7.6%), C. sunki Hort.

Ex Tanaka (n = 1; 3.8%), C. aurantium (n = 1; 3.8%), C. mitis

(n = 1; 3.8%), C. limon (n = 1; 3.8%), C. aurantiifolia (n = 1;

3.8%), C. ichangensis (n = 1; 3.8%), Poncirus trifoliata x Citrus

sinensis (n = 1; 3.8%), and C. changshan-huyou (n = 1; 3.8%).

Among the Citrus species used in the preclinical studies, there

was a predominance of six hybrid species in eight studies,

followed by three orange species in eight studies and three

types of lemons in four publications and tangerine species in

four articles. In the clinical studies, on the other hand, there is

a predominance of orange-based bergamot products (C.

bergamia; n = 3 studies) and a study with supplements

containing grapefruit (C. paradise).

\From these species, hydroalcoholic extracts or organic

fractions (n = 20; 86.9%), aqueous extract (n = 1; 4.3%),

and processed fruits (n = 3; 13.0%) were used, which were

incorporated to the diet (n = 14; 60.8%) or administered orally

by gavage (n = 9; 40.9%). In the clinical trials as a whole,

supplementation with encapsulated dry extract was used or

inclusion in the diet. In addition, 21 studies (80.7%) evaluated

the

chemical

composition

of

the

extracts,

with

predominance of compounds belonging to the class of^a

flavonoids,

such

as

naringin,

hesperidin,

neoeriocitrin,

neohesperidin,

nobiletin,

tangeretin,

and

naringenin

(Figure 2).

As

observed

in

Table

1,

the

method

of

inducing

hyperlipidemia in the preclinical studies was by cholesterol-

rich diet or cafeteria-type diet, conducted with rats (n = 12;

52.1%), mice (n = 8; 34.7%), and hamsters (n = 3; 13.0%). Among

the randomized clinical trials (Table 3), the clinical conditions of

the participants were in their entirety dyslipidemia (n = 4; 100%),

associated or not with coronary disease (n = 1, 25%), and

hypertension and glucose intolerance (n = 1; 25%). In the

preclinical and clinical studies, the outcomes evaluated were

the levels of total cholesterol (TC, n = 18; 100%), HDL (n

= 14; 77.7%), LDL (n = 12; 66.7%), VLDL (n = 2; 13.3%), IDL

(n = 1; 5.5%), and triglycerides (TG, n = 17; 94.4%).

From the analysis of the preclinical and clinical studies (Tables

2–4), it was found that the Citrus species were able to significantly

alter the lipid profile in the 26 (100%) studies, decreasing serum

total cholesterol (n = 25; 96.1%), LDL (n = 14; 53.8%),

triglycerides (n = 17; 65.3%), and VLDL (n = 2; 7.6%) and

increasing HDL (n = 4; 15.3%). In the liver, Citrus also

reduced TC and TG (n = 6; 23.0%), lipid accumulation (n =

5; 19.2%), and weight (n = 2; 7.6%). These effects were

accompanied by the maintenance (n = 1; 3.8%) of glutamic-

oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase

(GPT), and alkaline phosphatase (ALP) serum levels or the

FIGURE 1 | Flowchart of the studies included in the qualitative and quantitative synthesis.

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia

TABLE 1 | Detailed description of the preclinical studies of the effect of Citrus extract on hyperlipidemia included in the systematic review.

References,

country

Extract, plant

part, and

species

Composition

Model

Evaluated

parameters

Treatment protocol

Animal

(n/group)

Vinson et al.,

Hydroalcoholic extract

of whole dried ripe

fruits C. aurantium

25.7% ascorbic acid

Hamster fed on a high-

cholesterol diet

LDL, VLDL

Feed containing 3% of

the extract or 4% of

the extract associated

with ascorbic acid

(57 mmol/kg diet)

daily, for 4 or

10 weeks

Male

1998 (Vinson

et al., 1998)

9.9% flavonoids (quercetin,

hesperidin, naringenin, and

myricetin)

HDL, TC, TG, foam cell

injury

Golden

Syrian

EUA

31.2% protein

in the aorta artery

Hamsters

(n = 10)

3.2% ash

lipid peroxidation

30% carbohydrates

Bok et al.,

Hydroalcoholic extract

of the peel C. reticulata

2.7 g of protein

Rats fed on a high-

cholesterol diet

Plasmatic and hepatic

TC, TG, HDL, LDL

16.7 g/100 g of diet

for 6 weeks

Male

1999 (Bok et al.,

1999)

1.8 g of fat

AI^a, fecal neutral sterols,

HMGR, and ACAT

activities in liver tissue

Sprague

Dawley rats

(n = 10)

Korea

1.0 g of ash

20 g of fructose

16.5 g of glucose

8.6 g of sucrose

0.6 g of hesperidin

0.03 g of naringin and 9.67 g

of other sugars

Terpstra et al.,

2002 (Terpstra

et al., 2002)

Peels or waste stream

material of C. limon

-

Hamster fed on a high-

cholesterol diet

BW, FI, and liver weight

Diets containing 3% of

cellulose or lemon

peels or the waste

stream of the lemon

pectin extraction

Male hybrid

Netherlands

TC of plasma and liver

for 8 weeks

F1B Golden

Plasmatic TG, LDL,

HDL, VLDL

Syrian

bile acids, and fecal

sterols

Hamster

(n = 14)

Mollace et al.,

2011 (Mollace

et al., 2011)

Polyphenolic fraction

of C. bergamia Risso &

Poiteau peeled-off

fruits

Neoeriocitrin (77,700 ppm),

naringin (63,011 ppm),

neohesperidin

(72,056 ppm), melitidine

(15,606 ppm), and

brutieridine (33,202 ppm)

High-cholesterol diet-

induced hyperlipemia

BW, TC, LDL, HDL

10 or 20 mg/kg

daily (p.o.)

Male

Italy

TG and glucose

for 30 days

Wistar

Neutral sterols and fecal

bile acids

Rats

(n = 10)

Zulkhairi et al.,

(Zulkhairi et al.,

2010)

Aqueous extract (5%

and 10%) of dried

whole fruits C. mitis

Phenolic compounds

Rats fed on a high-

cholesterol diet

BW, TC, HDL, LDL, TG,

AI^b, sdLDL^c

5 mg/kg of extract at

5% and 10%

Male

Malaysia

Scavenging activity of

DPPH radicals, reducing

power, lipid

peroxidation (in vitro)

daily (p.o.)

Sprague

Dawley rats

(n = 6)

for 10 weeks

Ding et al.,

Hydroalcoholic extract

of C. ichangensis peel

Naringin, hesperidin,

poncirin, neoeriocitrin

High-fat diet-induced

BWG, FI

Diet supplemented

with 1% of extract, for

8 weeks

Female

2012 (Ding et al.,

2012)

narirutin, neohesperidin,

naringenin, nobiletin,

Obese

TC, TG, LDL, HDL, and

glucose

C57BL/6

mice (n = 7)

China

and tangeretin

Fecal and hepatic TC

and TG; size of EWAT;

mRNA expression of

PPARγ, LXR, and them

target genes in liver

tissue

Kang et al.,

Hydroalcoholic extract

of C

Tangeretin (55.13 mg/g)

High-fat diet-induced

BWG, FI

150 mg/kg/day of

extract (p.o.)

Male

2012 (Kang et al.,

2012)

sunki peel

Nobiletin (38.83 mg/g)

Obese

TC, TG, GPT, GOT, and

LDH, EPAT weight, liver

fat; p-AMPK, p-ACC,

and adiponectin mRNA

expression in EAT.

for 70 days

C57BL/6

mice

(n = 10)

(Continued on following page)

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Citrus Extract as a Perspective for the Control of Dyslipidemia

TABLE 1 | (Continued) Detailed description of the preclinical studies of the effect of Citrus extract on hyperlipidemia included in the systematic review.

References,

country

Extract, plant

part, and

species

Composition

Model

Evaluated

parameters

Treatment protocol

Animal

(n/group)

Korea

Hesperidin (17.11 mg/g)

In mature 3T3-L1

adipocytes: LKB1,

AMPK, ACC, PKA, and

HSL phosphorylation,

CPT-1a gene

expression, and glycerol

release

Rutin (17.02 mg/g)

Sinensetin (4.23 mg/g)

Raasmaja et al.,

2013 (Raasmaja

et al., 2013)

Hydroalcoholic extract

of C. grandis (L.)

Osbeck whole fruits

Naringin at 19%

High-fat diet-induced

BWG, FI

300, 600, or

1,200 mg/kg (p.o.)

daily

Female

Finland

Obese

TG, TC, HDL, glucose,

insulin, ghrelin, GLP-1

for 12 weeks

Zucker

PYY, leptin, and amylin

in plasma

Rats

(n = 10)

Lu et al.,

Hydroalcoholic extract

of Citrange (Poncirus

trifoliata x C. sinensis)

peel or flesh and seed

Bark extract

High-fat diet-induced

obese

BWG, FI, ipGTT, blood

glucose, serum TG, TC,

LDL and HDL, hepatic

TG and TC

Diet supplemented

with 1% w/w of peel

extract

Female

2013 (Lu et al.,

2013)

Neoeriocitrin (14.5 mg/g),

naringin (8.12 mg/g),

neohesperidin (21.1 mg/g),

and poncirin (14.1 mg/g)

Fecal TC and TG,

histological analysis

or 1% w/w of flesh

and seed

C57BL/6

mice (n = 6)

China

Seed extract

of liver tissue

extract, daily

Poncirin (4.85 mg/g)

Neohesperidin (1.87 mg/g)

Naringin (0.87 mg/g)

mRNA levels of PPARγ,

LXR, and their target

genes in liver tissue

for 8 weeks

Kim et al.,

Hydroalcoholic extract

of C. junos Tanaka

peel

Hesperidin (36.3 mg/100 g)

High-fat diet-induced

obese

BWG, FI

Diet supplemented

with 1% and 5% of

extract

Male

2013 (Kim et al.,

2013)

Naringin (11.6 mg/100 g)

TC, TG, glucose, insulin,

leptin, resistin, GOT,

GPT, histological

analysis of liver tissue

for 9 weeks

C57BL/6 J

mice (n = 8)

Korea

Rutin (2.7 mg/100 g)

AMPK phosphorylation

in muscle tissue

Quercetin (1.7 mg/100 g)

and tangeretin (0.7 mg/

100 g)

AMPK and PPARγ

activation in C2C12 and

HEK293 cells,

respectively

Muhtadi et al.,

2015 (Muhtadi

et al., 2015)

Hydroalcoholic extract

of C. sinensis fruit peel

-

High-fat diet-induced

hypercholesterolemia

TC; glucose in rats

125, 250, and

500 mg/kg (p.o.), daily

for 2 weeks

Male

Indonesia

induced by alloxan

monohydrate

After 4-week diet

Wistar rats

(n = 5)

Dinesh and

Hegde, 2016

(Dinesh and

Hegde, 2016)

Hydroalcoholic extract

of C. maxima leaves

Flavonoids, alkaloids,

carbohydrates, glycosides,

saponins, and tannins

Cafeteria diet and

Olanzapine-induced

obesity

BWG, FI

200 and 400 mg/kg

(p.o.), daily for

4 weeks

Female

India

TC, TG, HDL, LDL,

VLDL, GOT, GPT,

glucose

Wistar rats

(n = 6)

Liver weight and TG

Shin et al.,

Hydroalcoholic extract

of C. junos Tanaka

peel

-

Mice fed on a high-

cholesterol diet

BWG, FI

Diet supplemented

with 1% and 5% of the

extract

Male

2016 (Shin et al.,

2016)

TG, TC, HDL, GOT,

GPT, ALP, histological

analysis

for 10 weeks

C57BL/6 J

mice (n = 8)

Korea

of liver tissue

(Continued on following page)

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia

TABLE 1 | (Continued) Detailed description of the preclinical studies of the effect of Citrus extract on hyperlipidemia included in the systematic review.

References,

country

Extract, plant

part, and

species

Composition

Model

Evaluated

parameters

Treatment protocol

Animal

(n/group)

Expression of PPARα,

FAS, and HMGR in liver

tissue

Lipid accumulation and

expression of p-AMPK,

p-ACC, PPARα, CPT-1,

and HMGR in HepG2

cells

Ashraf et al.,

Hydroalcoholic extract

of C. sinensis peel

-

Rats fed on high-glucose

or cholesterol-rich diet

BWG, FI

Diet supplemented

with 10% Citrus peel

powder (functional)

and 5% peel extract

(nutraceutical), for

8 weeks

Male

2017 (Ashraf

et al., 2017)

TG, TC, LDL, HDL,

glucose, insulin

Sprague

Pakistan

Dawley rats

(n = 6)

Fayek et al.,

Methanolic extract,

hexanic extract,

aqueous homogenate

of C. reticulata

(Mandarin), C. sinensis

(sweet orange), C.

paradise (white

grapefruit), or C.

aurantiifolia (lime) fruit

peels

Nobiletin (%) in hexanic

extracts

Hypercholesterolemia

induced by diet rich in

cholesterol and bile salts

TC

0.1 ml of the

corresponding extract

(p.o.) for 8 weeks

Male

2017 (Fayek

et al., 2017)

Mandarin (10.14%)

TG and glucose

Wistar rats

(n = 6)

Egypt

Sweet orange (3.6%)

White grapefruit (0.9%)

Lime (0.0045%)

Pectin (%) in peel powder

Sweet orange (21.33%)

Lime (19.7%)

While grapefruit (11.66%)

Mandarin (9.14%)

Chou et al., 2018

(Chou et al.,

2018)

Methanolic extract of

C. reticulata

Narirutin (4.52 ± 0.31 mg/g),

hesperidin (9.14 ± 0.32 mg/

g), nobiletin (2.54 ±

0.07 mg/g)

High-fat diet-induced

AST, ALT, triglyceride,

total cholesterol,

glucose, insulin,

HOMA-IR

1% of the

corresponding extract

for 11 weeks

Male

China

Tangeretin (1.67 ±

0.05 mg/g)

obese

C57BL/6 J

mice (n = 8)

Feksa et al., 2018

(Feksa et al.,

2018)

Hydroalcoholic extract

of leaves of C. maxima

Gallic acid, catechin, caffeic

acid, epicatechin, rutin and

isoquercetin, and the major

compounds

High-fat diet and fructose

Blood count, AST, ALT,

triglyceride, total

cholesterol, LDL, HDL,

glucose, urea,

creatinine,

50 mg/kg

Male

Brazil

were caffeic acid (3.71 mg/g)

and catechin (3.65 mg/g

Wistar rats

(n =

Mir et al., 2019

(Mir et al., 2019)

Hydroalcoholic extract

of C. latifolia

-

Hypercholesterolemia

induced by diet rich in

cholesterol

triglyceride, and total

cholesterol

1% of the

corresponding extract

for 4 weeks

Male

Algeria

Wistar rats

(n = 10)

Sato et al., 2019

(Sato et al., 2019)

C. tumida peel powder

Calorie (275 kcal), moisture

(2.9 g), protein (7.4 g), fat

(2.7 g), ash (4.9 g),

carbohydrate (82.1 g), sugar

(28.4 g), fiber (53.7 g),

galacturonic acid (12.2 g),

and sodium (4.3 mg)

High-fat diet

AST, ALT, triglyceride,

total cholesterol, HDL-

C, creatinine, albumin,

calcium, and LDH

C. tumida peel

powder 5% (w/w)

Male

Japan

C57BL/6 J

mice (n = 8)

Tamaru et al.,

2019

(Hase-Tamaru

et al., 2019)

C. unshiu MARC

lyophilized and

powdered

76.1 g carbohydrate, 7.6 g

crude protein, 0.7 g crude

fat, 2.7 g ash, 12.9 g

moisture, 40.9 g

High-fat diet

Total cholesterol,

triglycerides, free fatty

acids, glucose, insulin,

and leptin

2.5%

Sprague

Dawley (SD)

rats (n = 7)

Japan

total fiber, 6.6 g total pectin,

14.4 g hesperidin, and 3.0 g

narirutin

5.0%, or 10.0%

Lee et al., 2020

(Lee et al., 2020)

C. unshiu: dried

extract (CPEW) and

lyophilized (CPEF)

Hesperidin, narirutin, and

synephrine

High-fat diet

AST, ALT, triglyceride,

total cholesterol, and

LDL-C

CPEW: 50 mg/kg;

100 mg/kg

Male

Korea

CPEF: 50 mg/kg;

100 mg/kg

SD rats

(n = 8)

Ling et al., 2020

(Ling et al., 2020)

C. changshan-huyou

Naringin, narirutin, and

neohesperidin

High-fat diet

PTFC: 25 mg/kg;

50 mg/kg; 100 mg/kg

(Continued on following page)

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia

reduction of GOT, GPT (n = 2; 7.6%), and lactate dehydrogenase

(LDH) (n = 1; 3.8%).

In addition, some Citrus products also reduced body weight

gain (BWG; n = 7; 26.9%), food intake (FI; n = 1; 3.8%), and lipid

accumulation in adipose tissue or cells (n = 3; 11.5%). In human, a

study also demonstrated their effect on the reduction of waist

circumference (WC), waist-to-hip ratio (WHR), and body mass

index (BMI). Taken together, these effects can reduce the risk of

atherosclerosis as shown in three studies (16.6%). However, its

effects on the lipid excretion are still controversial, since two

studies (11.0%) demonstrate increased excretion, two studies

(11.0%) did not identify changes, and only one study (5.5%)

found a reduction in excretion (Table 3). In parallel, some

authors investigated the effect of Citrus-based products on

glucose and their effects on blood glucose reduction (n = 8;

44.4%), insulin increase (n = 2; 11.0%), and glucose uptake in the

cell (n = 1; 5.5%).

In addition, several targets involved in the energy and nutrient

metabolism have been studied. As can be seen in Table 3, some

species

of

Citrus

demonstrated

effects

on

peroxisome

proliferator-activated receptor γ (PPARγ) and peroxisome

proliferator-activated receptor α (PPARα), downmodulating

fatty

acid

synthase

(FAS),

acyl-CoA

oxidase

(ACO),

uncoupling

protein

2

(UCP2),

and

adipocyte

fatty-acid-

binding protein (aP2), besides upregulating CD36 and acetyl-

CoA carboxylase (ACC). They can also act on liver X receptor

(LXR), reducing lipoprotein lipase (LPL), apolipoprotein E

(ApoE),

and

cholesterol

7α-hydroxylase

(CYP7A1)

and

increasing ATP-binding cassette transporter G1 (ABCG1) and

ATP-binding cassette transporter A1 (ABCA1).

The adiponectin signaling pathway also can be involved in the

lipid control. In fact, some Citrus products were able to increase

adiponectin; stimulate the phosphorylation of LKB1, AMP-

activated

protein

kinase

(AMPK),

ACC,

and

carnitine

palmitoyl transferase-1 (CPT-1); and reduce HMGR and

ACAT activities. Their effects on lipolysis were also observed

by the upmodulation of cAMP-dependent protein kinase (PKA)

and hormone-sensitive lipase (HSL), with increase in glycerol.

Besides adiponectin, Citrus seems to act reducing other

adipocytokines, as leptin and resistin, which regulate the

appetite and glucose metabolism and have been associated

with insulin resistance. Their effects were also observed in the

hormones involved with satiety and hunger control, as leptin,

glucagon-like peptide-1 (GLP-1), and ghrelin. Finally, the

antioxidant potential of Citrus has also been demonstrated,

which can offer benefits in reducing lipid oxidation and in the

development of atheromatous plaques.

Methodological Quality/Risk of Bias

The 23 preclinical studies, using the criteria provided by the

ARRIVE guidelines, were analyzed for methodological quality.

The studies showed a percentage of adequacy varying between 50

and 92% (83.82 ± 10.77%), with a greater weakness in the quality

of the methodological description of the studies (Supplementary

Table S2).

As for the clinical studies included in this research and

evaluated by the Cochrane list (Figure 3), all of them had

blinding outcome evaluators and incomplete outcomes. In

addition, 50% of the articles presented low risk of uncertain

bias regarding the criteria of generating a random sequence,

concealment

of

allocation,

blinding

of

the

participants,

reporting of the selective outcome, and other sources of bias

(conflict of interest, based on the source of funding for the study

and method of determination of the sample size).

Meta-Analysis

For the meta-analysis, the preclinical studies measured the level of

total cholesterol [n = 23; 100%; I² = 99.1% (98.9%; 99.2%)],

triglycerides [n = 20; 87%; I² = 99.4% (99.3%; 99.5%)], LDL [n =

12; 52.2%; I² = 99.1% (98.9%; 99.3%)], and HDL [n = 14; 60.9%; I²

= 93.4% (90.6%; 95.4%)]. As for the clinical studies, three clinical

trials with 92, 98, and 237 participants were included in the

TABLE 1 | (Continued) Detailed description of the preclinical studies of the effect of Citrus extract on hyperlipidemia included in the systematic review.

References,

country

Extract, plant

part, and

species

Composition

Model

Evaluated

parameters

Treatment protocol

Animal

(n/group)

AST, ALT, triglyceride,

total cholesterol, LDL-C,

and HDL-C

Golden

hamsters

(n = 12)

China

Ke et al., 2020

(Ke et al., 2020)

C. reticulata Blanco

Nobiletin (98.34 mg/g),

heptamethoxyflavone

(44.26 mg/g), tangeretin

(26.20 mg/g), and

isosinensetin (26.14 mg/g)

High-fat diet

Triglyceride, total

cholesterol, LDL-C, and

HDL-C

0.2 and 0.5% JZE

C57BL/6 J

mice (n = 8)

China

glutamicp.o., intragastric gavage; TC, total cholesterol; TG, triglycerides; LDL, low-density lipoprotein; HDL, high-density lipoprotein; VLDL, very low-density lipoprotein; LDH, lactate

dehydrogenase; GOT, -oxaloacetic transaminase; GPT, glutamic-pyruvic transaminase; EWAT, epididymal white adipose tissue; PPARγ, peroxisome proliferator-activated receptor γ;

FAS, fatty acid synthase; ACO, acyl-CoA oxidase; LXRα, liver X receptor α; LXRβ, liver X receptor β; AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase; PKA, cAMP-

dependent protein kinase; HSL, hormone-sensitive lipase. GLP-1, glucagon-like peptide-1; PYY, pancreatic peptide YY; BWG, body weight gain; FI, food intake; ipGTT, intraperitoneal

glucose tolerance test; ALP, alkaline phosphatase; FAS, fatty acid synthase receptor; CPT-1, carnitine palmitoyl transferase-1; HMGR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase;

EPAT, epididymal and perirenal adipose tissue; EAT, epididymal adipose tissue.

aThe duration of the experiment is not explicitly informed in the article. AI, atherogenic index.

b[(TC-HDL)/HDL].

c(LDL/HDL); sdLDL, small dense LDL, particle size.

d(TG/HDL).

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia

TABLE 2 | Outcomes of the preclinical studies included in this systematic review.

Reference

Experimental group (mmol/L)

Control group (mmol/L)

Summary of results

Vinson et al., (Vinson et al.,

1998)

Baseline: TC: 5.84; HDL: 3.31;

TG: 25.1

Baseline: TC: 10.3; HDL: 2.84;

TG: 41.6

↓TC and TG

10 weeks: TC: 6.88; HDL: 1.68;

TG: 27.1

10 weeks: TC: 15.1; HDL: 1.48;

TG: 55.9

↓ lipid peroxidation

↓ atherosclerosis signals (↓ area and density of foam cells),

without changing BW

Bok et al. (Bok et al., 1999)

Baseline

Baseline:

↓ plasma TC

6 weeks: TC: 2.44; HDL: 0.61;

TG: 1.22

6 weeks: TC: 3.8; HDL: 0.57;

TG: 1.12

↓ hepatic TC and TG, without changing HDL, TG, and LDL

plasmatic

↓AI and cholesterol excretion

↓ HMGR and ACAT activities

Terpstra et al. (Terpstra et al.,

2002)

Baseline:

Baseline:

↓ plasma and liver TC, ↓ VLDL + LDL being more effective in

↓VLDL, without changing HDL, ↑ excretion of fecal neutral

sterols and bile acids

8 weeks (lemon peel): TC: 3.51

8 weeks (cellulose): TC: 4.21

without changing BW, FI, and liver weight

8 weeks (waste stream): TC: 3.44

Mollace et al. (Mollace et al.,

2011)

Baseline:

Baseline:

↓TC, LDL, and TG, without changing BW, HDL and glucose

30 days (10 mg): TC: 5.95; LDL: 4.49;

HDL: 0.58; TG: 2.75

30 days: TC: 8.19; LDL: 6.04;

HDL: 0.53; TG: 2.74

↑ fecal neutral sterols and bile acids

30 days (20 mg): TC: 5.00; LDL: 3.90;

HDL: 0.65; TG: 2.74

Zulkhairi et al. (Zulkhairi et al.,

2010)

Baseline (5%)

Baseline: TC: 1.75; LDL: 0.45;

HDL: 0.85; TG: 0.54

↓ TC, LDL, TG

TC: 1.73; LDL: 0.45; HDL: 1.34;

TG: 0.76

4 weeks

↑ HDL

Baseline (10%)

TC: 2.13; LDL: 0.93; HDL: 0.89;

TG: 0.79

↓AI and sdLDL

TC: 1.68; LDL: 0.49; HDL: 1.27;

TG: 0.74

Antioxidant activity, without changing BW

4 weeks (5%)

TC: 1.28; LDL: 0.27; HDL: 1.39;

TG: 0.63

4 weeks (10%)

TC: 1.06; LDL: 0.23; HDL: 1.54;

TG: 0.53

Ding et al. (Ding et al., 2012)

Baseline:

Baseline:

↓ BWG

8 weeks

8 weeks

↓TC and LDL plasmatic

TC: 2.27; LDL: 0.35; HDL: 2.32;

TG: 0.70

TC: 2.65; LDL: 0.46; HDL: 1.95;

TG: 0.70

↓ hepatic TC, TG, glucose, and adipocyte size, without

changing

Plasmatic FI, HDL, and TG and

fecal TC and TG

↓ expression of PPARγ (↓FAS, ACO, and UCP2 and ↑ CD36) ↓

LXR α and β (↓ ApoE, CYP7A1, LPL, and ↑ABCA1)

Kang et al. (Kang et al., 2012)

Baseline:

Baseline:

↓ BWG without changing in FI

70 days

70 days: TC: 4.63; TG: 1.56

↓ TC, TG, LDH, GOT, and GPT

TC: 3.81; TG: 0.94

↓ weight and cell size of EPAT

↓ liver fat

↑ p-AMPK, p-ACC, p-LKB1, and adiponectin

↑ glycerol release

↑ p-PKA and p-HSL

Raasmaja et al. (Raasmaja

et al., 2013)

Baseline (300 mg/kg)

Baseline

Tendency to ↓ TC, glucose, and TG and ↑ HDL

TC: 3.72; HDL: 1.42; TG: 8.34

TC: 3.56; HDL: 1.67; TG: 7.31

↓ GLP-1 and reversing the ↓ of ghrelin, without changing

BWG, FI

Baseline (600 mg/kg)

12 weeks

PYY, leptin, insulin, and amylin

TC: 3.13; HDL: 1.70; TG: 6.27

TC: 4.13; HDL: 0.52; TG: 15.76

Baseline (1,200 mg/kg)

TC: 3.59; HDL: 1.53; TG: 8.11

12 weeks (300 mg/kg)

TC: 4.23; HDL: 0.44; TG: 16.68

12 weeks (600 mg/kg)

TC: 3.62; HDL: 0.80; TG: 12.57

12 weeks (1,200 mg/kg)

TC: 4.36; HDL: 0.80; TG: 17.42

Lu et al. (Lu et al., 2013)

Baseline

Baseline

↓ BWG

(Continued on following page)

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia

TABLE 2 | (Continued) Outcomes of the preclinical studies included in this systematic review.

Reference

Experimental group (mmol/L)

Control group (mmol/L)

Summary of results

8 weeks (peel)

8 weeks

Improves glucose tolerance and insulin resistance

TC: 2.30; LDL: 0.36; HDL: 2.00;

TG: 0.70

TC: 2.64; LDL: 0.41; HDL: 1.97;

TG: 0.70

↓ serum glucose, TC, and LDL

8 weeks (seed)

↓ hepatic TC and TG, without changing FI, serum HDL, and

fecal TC and TG

TC: 2.43; LDL: 0.41; HDL: 1.87;

TG: 0.74

↓ PPARγ (↓ ap2, FAS); ↓ LXRβ (↓ LPL and ApoE and ↑ ABCG1)

↓ lipid accumulation in liver tissue

Kim et al. (Kim et al., 2013)

Baseline

Baseline:

↓ BWG, glucose, TG, TC, insulin, leptin, and resistin

9 weeks (1%)

9 weeks

↑ glucose uptake

TC: 2.00; TG: 0.85

TC: 2.37; TG: 0.88

↓ liver tissue fat

9 weeks (5%)

↑ PPARγ and AMPK, without changing FI, GOT, and GPT

TC: 1.91; TG: 0.76

Muhtadi et al. (Muhtadi et al.,

2015)

Baseline (125 mg/kg): TC: 4.31

Baseline: TC: 3.77

↓ TC and glucose

Baseline (250 mg/kg): TC: 5.08

2 weeks: TC: 3.27

Baseline 500 (mg/kg): TC: 4.87

2 weeks (125 mg/kg): TC: 1.88

2 weeks (250 mg/kg): TC: 2.13

2 weeks (500 mg/kg): TC: 2.02

Dinesh and Hegde (Dinesh

and Hegde, 2016)

Baseline

Baseline:

↓ BWG and FI

4 weeks (200 mg/kg)

4 weeks

↓ TC, TG, LDL, and VLDL

TC: 79.76; LDL: 54.31; HDL: 40.68;

TG: 104.3

TC: 88.75; LDL: 74.71; HDL:

35.11; TG: 130.0

↑ HDL

4 weeks (400 mg/kg)

↓ GOT and GPT

TC: 75.77; LDL: 51.75; HDL: 43.22;

TG: 98.05

↓ liver weight and TG

↓ glucose

Shin et al. (Shin et al., 2016)

Baseline:

Baseline:

↓ BWG

10 weeks (1%)

10 weeks

↓ TC, LDL, GOT, GPT, ALP, without changing FI, HDL

TC: 2.89; LDL: 1.81; HDL: 0.87

TC: 4.03; LDL: 3.03; HDL: 0.80

↓ liver fat content and weight

10 weeks (5%)

↑ p-AMPK, p-ACC, PPARα, and CPT-1 expression

TC: 2.96; LDL: 1.80; HDL: 0.80

↓ FAS and HMGR expression

↓ lipid accumulation

Ashraf et al. (Ashraf et al.,

2017)

Baseline (powder)

Baseline

Tendency to

TC: 3.34; HDL: 1.19; LDL: 1.67;

TRI: 1.07

TC: 3.30; HDL: 1.17; LDL: 1.63;

TRI: 1.04

↓ BWG and FI

Baseline (extract)

8 weeks

↓ TG, TC, and LDL

TC: 3.32; HDL: 1.21; LDL: 1.62;

TRI: 1.05

TC: 3.81; HDL: 1.17; LDL: 1.85;

TRI: 1.16

↑ HDL

8 weeks (powder)

↓ glucose and ↑ insulin

TC: 3.14; HDL: 1.21; LDL: 1.52;

TRI: 1.01

8 weeks (extract)

TC: 3.03; HDL: 1.24; LDL: 1.44;

TRI: 0.97

Fayek et al. (Fayek et al.,

2017)

Baseline:

Baseline:

Tendency to ↓ TC

Tangerine (alcoholic extract)

Diet

↓ TG and glucose

TC: 2.00; TG: 0.78

TC: 3.92; TG: 2.66

Orange (alcoholic extract)

TC: 3.25; TG: 0.94

Hybrid (alcoholic extract)

TC: 3.95; TG: 0.85

Lime (alcoholic extract)

TC: 5.47; TG: 0.51

Chou et al. (Chou et al., 2018)

Baseline:

Baseline:

Tendency to ↓ TC

11 weeks (1%)

11 weeks (diet)

↓ TG and insulin resistance

TC: 3.85; TG: 0.44

TC: 4.68; TG: 0.85

Feksa et al. (Feksa et al.,

2018)

Baseline

Baseline:

Tendency to

45 days (50 mg/kg)

45 days (diet): TC: 3.34; TG:

3.38; HDL: 0.47; LDL: 1.23

↓ TG, TC, and LDL

TC: 2.12; TG: 2.84; HDL: 0.34;

LDL: 0.61

Mir et al. (Mir et al., 2019)

Baseline

Baseline:

Tendency to

4 weeks (1%)

4 weeks (diet)

↓ TG and TC

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia

quantitative analyses, which were performed with patients with

dyslipidemia and demonstrated the Citrus effects on the levels of

total cholesterol [I² = 94.5% (87.3%; 97.6%)], triglycerides [I²

= 95.6% (90.5%; 98.0%)], LDL [I² = 96.6% (93.0%; 98.4%)], and

HDL [I² = 81.4% (42.2%; 94.0%)] (in both, n = 3; 100%).

The presentation of the forest graphs was distributed

according to the results of the levels of total cholesterol,

triglycerides, LDL, and HDL for preclinical and clinical

studies. Through the global analysis of preclinical studies, a

reduction of −1.08 mmol/L (95% CI: 1.23; −0.92; Figure 4A)

was found in total cholesterol, equivalent to 41.76 mg/dL; a

reduction of −0.50 mmol/L (95% CI: 0.69; −0.31; Figure 4B)

was found in triglycerides, corresponding to 44.28 mg/dL; and a

reduction of −0.71 mmol/L (95% CI: 0.97; −0.45; Figure 4C) was

found in LDL, what represents 27.45 mg/dL. In addition, an

increase of 0.11 mmol/L in the HDL levels was verified (95%

CI: 0.05; 0.17; Figure 4D), equivalent to 4.25 mg/dL.

As illustrated in Figure 5, in the studies carried out on

humans, the levels (mg/dL) of total cholesterol (MD = −42.03,

95% CI: 73.53; −10.52), triglycerides (MD = −62.41, 95% CI:

110.09; −14.73), and LDL (MD = −37.76, 95% CI: 69.45; −6.06)

were reduced after treating patients with Citrus extracts. In

addition, it was observed that these patients had increased

HDL levels (MD = 5.85, 95% CI: 0.41; 11.28). Although a high

heterogeneity has been observed (I² > 75%), the synthase of the

results obtained with individual studies favors treatment to the

control of serum lipids. After the analysis of subgroups, high

heterogeneity was still verified and the sensitivity analysis did not

change the result of the general analysis (data not shown).

DISCUSSION

This systematic review compiled data from 25 studies on the

effects of Citrus-based products in the control of dyslipidemia.

Based on the countries where the studies were carried out, most of

them were developed in countries of Asia (such as Korea and

China) and the European Union, in addition to United States and

Egypt, which are among the biggest Citrus product makers in the

world (FAS, 2018). In fact, countries that have greater production

TABLE 2 | (Continued) Outcomes of the preclinical studies included in this systematic review.

Reference

Experimental group (mmol/L)

Control group (mmol/L)

Summary of results

TC: 3.8; TG: 0.9

TC: 5.9; TG: 1.8

Sato et al. (Sato et al., 2019)

Baseline:

Baseline:

Tendency to

4 weeks (5%)

4 weeks (diet)

↓ TG and TC

TC: 3.31; TG: 0.28; HDL: 2.06

TC: 4.39; TG: 0.41; HDL: 2.42

Tamaru et al. (Hase-Tamaru

et al., 2019)

Baseline:

Baseline:

Tendency to

4 weeks (2.5%)

4 weeks (diet)

↓ TG and TC

TC: 2.01; TG: 1.67

TC: 2.27 TG: 2.00

↓ free fatty acids, glucose, insulin, and leptin

4 weeks (5%)

↓ FAS, G6PDH in cytosol, and PAP in microsome

TC: 2.22; TG: 1.63

4 weeks (10%)

TC: 1.72; TG: 2.74

Lee et al. (Lee et al., 2020)

Baseline

Baseline:

Tendency to

8 weeks (CPEW 50 mg/kg): TC: 4.00;

TG: 2.89; LDL: 2.58

8 weeks (diet): TC: 4.00; TG:

2.89; LDL: 2.58

↓ TG and TC

8 weeks (CPEW 100 mg/kg): TC: 3.54;

TG: 2.52; LDL: 2.27

8 weeks (CPEF 50 mg/kg): TC: 4.08;

TG: 2.79; LDL: 2.56

8 weeks (CPEF 100 mg/kg): TC: 3.64;

TG: 2.59; LDL: 2.37

Ling et al. (Ling et al., 2020)

Baseline

Baseline:

Tendency to

4 weeks (25 mg/kg): TC: 32.00; TG:

10.20; HDL: 2.30; LDL: 11.41

4 weeks (diet)

↓ TG, TC, and LDL-C

4 weeks (50 mg/kg): TC: 22.30; TG:

5.30; HDL: 2.83; LDL: 9.83

TC: 41.59; TG: 11.15; HDL:

4.95; LDL: 11.80

4 weeks (100 mg/kg): TC: 21.70; TG:

5.30; HDL: 2.65; LDL: 8.67

Ke et al. (Ke et al., 2020)

Baseline

Baseline:

Tendency to

4 weeks (0.2%): TC: 5.69; TG: 0.28;

HDL: 4.10; LDL: 1.01

4 weeks (diet)

↓ TG, TC, and LDL-C

4 weeks (0.5%): TC: 5.04; TG: 0.28;

HDL: 3.84; LDL: 0.81

TC: 5.62; TG: 0.41; HDL: 4.20;

LDL: 1.20

TC, total cholesterol; TG, triglycerides; LDL, low-density lipoprotein; HDL, high-density lipoprotein; VLDL, very low-density lipoprotein; BW, body weight; HMGR, 3-hydroxy-3-

methylglutaryl-coenzyme A reductase; ACAT, acyl-CoA cholesterol acyltransferase; AI, atherogenic index; FI, food intake; BWG, body weight gain; PPARγ, peroxisome proliferator-

activated receptor γ; FAS, fatty acid synthase; ACO, acyl-CoA oxidase; UCP2, uncoupling protein 2; CD36, cluster of differentiation 36; LXR, liver X receptor; ApoE, apolipoprotein E;

CYP7A1, cholesterol 7α-hydroxylase; LPL, reducing lipoprotein lipase; ABCA1, ATP-binding cassette transporter A1; LDH, lactate dehydrogenase; GPT, glutamic-pyruvic transaminase;

GOT, glutamic-oxaloacetic transaminase; AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase; PKA; AMP-dependent protein kinase; HSL, hormone-sensitive lipase;

PYY, pancreatic peptide YY; GLP-1, glucagon-like peptide-1; ABCG1, ATP-binding cassette transporter G1; ALP, alkaline phosphatase; CPT-1, carnitine palmitoyl transferase-1; G6PDH,

glucose-6-phosphate dehydrogenase; PAP, phosphatidic acid phosphohydrolase in the microsome.

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia

of natural resources tend to explore their products more from a

commercial and scientific point of view.

Through the scientific analyses compiled, we can also verify

that species of the genus Citrus have the potential to reduce the

serum levels of total cholesterol (TC), triglycerides (TGs), LDL,

and VLDL and increase HDL. Consequently, Citrus-based

products reduced the body weight, lipid accumulation, and

atherosclerosis risk by the modulation of proteins and genes

involved in the lipid metabolism. Recently, a study with a

standardized extract containing Citrus sinensis L. Osbeck

associated with Citrus limon (Chiechio et al., 2021) also

demonstrated an effect in controlling the levels of total

cholesterol and triglycerides as well as glycemia, possibly due

to its composition rich in anthocyanins, flavonoids, and

hydroxycinnamic acids, reinstating the high potential of Citrus

species in lipid control.

These effects were studied mainly in the animal models of

dyslipidemia induced by cholesterol- or high-fat diets. In these

protocols, lipids ingested are initially degraded by intestinal lipase

and, in enterocytes, TGs are resynthesized and associated with

cholesterol and lipoproteins (ApoB-48, ApoE, and ApoC-II),

forming chylomicrons. These distributed fatty acids between

tissues and their remnants are metabolized in the liver. In this

organ, fatty acid and glucose activate metabolic pathways for

energy synthesis and storage, so that excess citrate is converted by

citrate lyase (ACLY) into acetyl-CoA, which by the action of

acetyl-CoA

carboxylase

(ACC)

forms

malonyl-CoA.

This

metabolic intermediate is used by the cell to produce fatty acid

through the action of the enzymes Stearoyl-CoA Desaturase-1

(SCD1)

and

fatty

acid

synthase

(FAS),

in

addition

to

downregulating CPT-1, an important transporter of Acil-Coa

into the mitochondria which enables its β-oxidation. These fatty

acids give rise to triglyceride molecules. In addition, acetyl-CoA

can participate in the synthetic pathway of cholesterol, forming

HMG-CoA which is converted into mevalonic acid by HMGR.

This originates the free cholesterol molecule, which can be

TABLE 3 | Detailed description of the clinical studies of the effect of Citrus extract on hyperlipidemia included in the systematic review.

References/

country

Extract, plant

part and

species

Composition

Sample

Pathology

Parameters

evaluated

Treatment protocol

Gorinstein et al.,

Fresh fruit peels of

red grapefruit or

blond grapefruit

processed

Anthocyanins

57 patients

(39–72 years)

Hypertriglyceridemia and

coronary

HR, BP, BW

Daily supplementation with

red or blond grapefruits

associated with anti-

atherosclerosis diet for

30 days (n = 19/group)

2007 (Gorinstein

et al., 2007)

Red: 51.5 mg/100 g

disease

CT, LDL, HDL,

Israel

Blond: 49.3 mg/100 g

TG, serum

antioxidant activity

by ABTS and

TEAC

Flavonoids (naringin)

Red: 21.61 mg/100 g

Blond: 19.53 mg/100 g

Total fibers

Red: 1.39 g/100 g

Blond: 1.37 g/100 g

Mollace et al.,

2011 (Mollace

et al., 2011)

Polyphenolic

fraction of C.

bergamia peeled-

off fruits

Neoeriocitrin (77,700 ppm)

237 patients

Hyperlipemia associated or

not with hyperglycaemia

TC, LDL, HDL,

500 or 1,000 mg/day

encapsulated with 50 mg

ascorbic acid, for 30 days

(n = 104–32/group)

Italy

Naringin (63,011 ppm)

TG, reactive

vasodilation

Neohesperidin

(72,056 ppm) and

melitidine (15,606 ppm)

Brutieridine (33,202 ppm)

Toth et al., 2016

(Toth et al.,

2015)

Bergavit

®

(Bergamot juice

derived extract, C.

bergamia)

150 mg of flavonoids

80 individuals

(42 men and

38 women)

Moderate

hypercholesterolemia

TC, LDL, HDL, TG,

VLDL, IDL, IMT,

LDL size

150 mg/day for 6 months

(n = 80)

Italy

16% of neoeriocitrin

47% neohesperidin

37% naringin

Cai et al., 2017

(Cai et al., 2017)

C. bergamia extract

(CitriCholess

®)

25% bioflavonoids, sterols

and orange oil (820 mg/

day), vitamin C (50 mg/

day), vitamin B6 (20 mg/

daily), B12 (2,000 µg/day),

and folic acid (800 µg/day)

98 older

people

Dyslipidemia and arterial

hypertension and problems

of glucose intolerance

TG, TC, LDL, HDL,

glucose, BW, WC,

HC, WHR,

and BMI

500 mg/day for 12 weeks

(n = 48–50/group)

China

Legend: TC, total cholesterol; TG, triglycerides; LDL, low-density lipoprotein; HDL, high-density lipoprotein; TEAC, Trolox-equivalent antioxidant capacity; HR, heart rate; BP, blood

pressure; BW, body weight; IMT, carotid intima-media thickness; BW, body weight (kg); WC, waist circumference (cm); HC, hip circumference (cm); WHR, waist-to-hip ratio; BMI, body

mass index.

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia

esterified by acyl-CoA:cholesterol acyltransferase (ACAT) or

converted into bile acids by CYP7A1. TG, free cholesterol, and

cholesterol ester conjugate with lipoproteins (ApoE, ApoC-II,

and ApoB-100) constituting the VLDL molecule (TGs >

cholesterol). This lipoprotein distributes fatty acids to tissues

by the action of lipoprotein lipase (LPL) and becomes IDL (TGs ≈

cholesterol, ApoB-100, ApoE) and later LDL (TGs, < cholesterol,

ApoB-100). That way, high-lipid diets increase the plasmatic

concentrations of TG, TC, VLDL, IDL, and LDL (DiNicolantonio

and O’Keefe, 2018; Andreadou et al., 2020). These mechanisms

can be observed in Figure 6 (black lines).

Through this review, it was found that the effect of Citrus-

based products on the release of adipocytokines and their

signaling pathways has been studied. These molecules are

produced by adipose tissue and control several metabolic

pathways, in addition to affecting the state of hunger and

TABLE 4 | Outcomes of the clinical studies included in this systematic review.

Reference

Experimental group

(mg/dL)

Control group (mg/dL)

Summary of results

Gorinstein et al. (Gorinstein et al.,

2007)

Baseline:

Baseline:

Red: ↓ TC, LDL, and TG

Red

TC: 306.26

Blond: ↓ LDL only

TC: 258.70

LDL: 243.23

Both: ↑ serum antioxidant activity, without change in HR,

BP, BW,

LDL: 193.73

HDL: 46.20

HDL

HDL: 52.59

TG: 205.49

TG: 149.68

Blond

TC: 283.06

LDL: 217.32

HDL: 50.27

TG: 193.97

Mollace et al. (Mollace et al., 2011)

Baseline (500 mg)

Treated with capsules containing

↓ TC, TG, and LDL

TC: 286.00

500 mg of maltodextrin and 50 mg of

ascorbic acid

↑ HDL

LDL: 184.96

Baseline

↓ glucose

HDL: 34.55

TC: 275.67

↑ reactive vasodilation

TG: 266.87

LDL: 186.31

Baseline (1,000 mg)

HDL: 34.59

TC: 279.40

TG: 275.62

LDL: 189.70

TC: 279.40

HDL: 32.78

LDL: 185.64

TG: 270.11

HDL: 35.05

After 30 days (500 mg)

TG: 275.71

TC: 211.42

LDL: 132.79

HDL: 40.53

TG: 180.18

After 30 days (1,000 mg)

TC: 201.99

LDL: 125.34

HDL: 46.00

TG: 157.48

Toth et al. (Toth et al., 2015)

Baseline

Baseline:

↓ TC, LDL, TG, and IMT

TC: 224.28

TC: 255.22

↑ HDL, IDL, and LDL size

LDL: 143.07

LDL: 177.88

without changing VLDL

HDL: 54.13

HDL: 50.27

TG: 132.86

TG: 159.43

Cai et al. (Cai et al., 2017)

Baseline

Baseline

↓ LDL

TC: 211.13

TC: 217.32; LDL: 138.43; HDL: 51.81; TG:

170.94

↓ BW, WC,

LDL: 131.09

TC: 210.36

WHR, and BMI

HDL: 49.88

LDL: 132.63

without changing TG, TC, HDL, glucose, HC

TG: 192.20

HDL: 52.20; TG: 172.71

500 mg

TC: 198.76

LDL: 121.03

HDL: 50.27

TG: 162.09

Legend: TC, total cholesterol; TG, triglycerides; LDL, low-density lipoprotein; HDL, high-density lipoprotein; TEAC, Trolox-equivalent antioxidant capacity; HR, heart rate; BP, blood

pressure; BW, body weight; IMT, carotid intima-media thickness; BW, body weight (kg); WC, waist circumference (cm); HC, hip circumference (cm); WHR, waist-to-hip ratio; BMI, body

mass index.

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia

satiety and being related to the development of coronary diseases

and metabolic disorders (Cao, 2014). Citrus products reduce

adiponectin (Kang et al., 2012), whose action on specific

receptors (AdipoR) increases the phosphorylation of LKB1 and

AMPK (Kang et al., 2012; Shin et al., 2016). It negatively

modulates ACC (Kang et al., 2012; Shin et al., 2016), reducing

malonyl-Coa levels and, consequently, increasing CPT-1 (Shin

et al., 2016); in addition, it decreases the HMGR activity (Bok

et al., 1999; Shin et al., 2016) and modulates genes like LXR (Ding

et al., 2012; Lu et al., 2013) and PPAR (Kim et al., 2013; Shin et al.,

2016; Lu et al., 2018). Through these genes, Citrus regulates

several protein targets involved in lipogenesis (FAS, aP2, ACC)

(Ding et al., 2012; Lu et al., 2013; Shin et al., 2016), lipoprotein

formation and metabolism (ApoE, LPL) (Ding et al., 2012; Lu

et al., 2013), cholesterol metabolism (CYP7A1) (Ding et al.,

2012), and cholesterol and lipid efflux (ABCG1 and ABCA1)

(Ding et al., 2012; Lu et al., 2013). At the same time, its ability to

stimulate the PKA-HSL pathway has also been observed (Kang

et al., 2012), increasing the degradation of TG in glycerol and fatty

acid, in addition to reducing the activity of ACAT (Bok et al.,

1999), which contributes to the reduction of cholesterol ester

levels. It is worth mentioning that bio-products based on Citrus

help in glycemic control (Mollace et al., 2011; Ding et al., 2012;

Kim et al., 2013; Lu et al., 2013; Raasmaja et al., 2013; Muhtadi

et al., 2015; Dinesh and Hegde, 2016; Ashraf et al., 2017; Fayek

et al., 2017), possibly by reducing resistin (Kim et al., 2013), an

adipocytokine whose increase has been associated with insulin

resistance, atherosclerosis, oxidative stress, and inflammation. All

of these molecular events result in decreased lipogenesis and

increased lipid oxidation, contributing to the control of the lipid

profile (Figure 6).

However, some results seem contradictory, such as the effect

of Citrus in reduction of the mRNA levels of PPARγ target genes,

including ACO and UCP2 in the liver tissue (Ding et al., 2012).

FIGURE 2 | Chemical structure of the main flavonoids found in Citrus.

FIGURE 3 | Methodological quality of clinical trials included.

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia

ACO is the first enzyme of peroxisomal β-oxidation which will

reduce the accumulation of lipids in the liver and promote its

excretion (Ferdinandusse et al., 2007). On the other hand, UCP2

is an uncoupling protein which acts as a carrier of protons present

in the inner membrane of mitochondria and contributes to

thermogenesis, being a positive factor for the prevention of

obesity (Brand and Esteves, 2005). Thus, upregulation of these

mRNAs would contribute to the observed outcomes. However,

the absence of baseline conditions for these targets makes it

difficult to understand these data, so further studies are needed to

elucidate this mechanism.

Similarly, Citrus seems to increase CD36 (Ding et al., 2012),

the fatty acid translocase protein that facilitates the transport of

fatty acids, the hepatic uptake of fatty acids, and the

accumulation of fat and has a high affinity for binding with

the oxidized LDL molecule, increasing the inflammatory activity

and

being

a

main

condition

for

the

development

of

atherosclerosis

and

thrombosis

(Pepino

et

al.,

2014).

However, the correlation with the observed outcomes also

needs to be further investigated, since the experimental

conditions of the study do not allow a thorough analysis of

this target in the experimental model used, as well as in the

primary outcome studied.

It is also worth noting that some studies have shown that

Citrus can help control hunger promoting the modulation of

ghrelin. Known as “Hunger Hormone,” this peptide is

produced by endocrine cells present in the stomach and

acts in the control of hunger, adiposity, and glucose- and

energy-homeostasis, among other functions (Pradhan et al.,

2013). More over, Citrus also downregulates leptin and GLP-1

levels, which are involved with satiety control. Leptin, a

hormone produced by adipose tissue, plays an important

role in the control of energy homeostasis, the excess and

resistance of which are associated with obesity, leading to

failures

in

the

signaling

mechanisms

associated

with

decreased nutrition and body weight control (Pan and

Myers, 2018). On the other hand, glucagon-like peptide 1

(GLP-1) is a gut hormone that promotes satiety; potentiates

insulin release and suppression of glucagon release in response

to nutrient intake; and decreases postprandial plasma levels of

glucose (Andersen et al., 2018). Thus, the effects observed for

Citrus in the reduction of GLP-1 may be related to overnight

fasting

or

long-term

regulation

of

eating

and

energy

metabolism, requiring further investigation.

The notations are as follows: ABCA1: ATP-binding cassette

transporter A1; ABCG1: ATP-binding cassette transporter G1;

ACAT: acyl-CoA:cholesterol acyltransferase; ACC: acetyl-

CoA

carboxylase;

ACLY:

citrate

lyase;

ACO:

acyl-CoA

oxidase;

AdipoR:

adiponectin

receptor;

AMPK:

AMP-

activated protein kinase; aP2: adipocyte fatty-acid-binding

protein;

ApoB-100:

apolipoprotein

B-100;

ApoC-II:

apolipoprotein C2; ApoE: apolipoprotein E; CD36: cluster

of differentiation 36; CPT-1: carnitine palmitoyl transferase-

1; CYP7A1: cholesterol 7α-hydroxylase; FAS: fatty acid

synthase;

GLUT

4:

glucose

transporter

4;

HMGR:

3-

hydroxy-3-methylglutaryl-coenzyme

A

reductase;

HSL:

FIGURE 4 | Forest plot of the preclinical studies that evaluated the effect

of Citrus species on total cholesterol (A), triglycerides (B), LDL (C), and HDL

(D) levels. The numbers on the x-axis indicate the effect of the treatment and

its favoring. SD: standard deviation of the differences. MD: difference

between the means.

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia

hormone-sensitive

lipase;

IDL:

intermediate

low-density

lipoprotein;

LDL:

low-density

lipoprotein;

LKB1:

liver

kinase B1; LPL: lipoprotein lipase; LXR: liver X receptor;

p-ACC:

phosphorylated

acetyl-CoA

carboxylase;

PKA:

cAMP-dependent

protein

kinase;

PPAR:

peroxisome

proliferator-activated

receptor;

SCD1:

Stearoyl-CoA

FIGURE 5 | Forest plot of the clinical studies that evaluated the effect of Citrus species on total cholesterol (A), triglyceride (B), LDL (C), and HDL (D) levels. The

numbers on the x-axis indicate the effect of the treatment and its favoring. SD: standard deviation of the differences. MD: difference between the means.

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia

Desaturase-1; TC: total cholesterol; TGs: triglycerides; UCP2:

uncoupling protein 2; VLDL: very low-density lipoprotein.

The effects of Citrus bioproducts on the lipid profile may be

related to the presence of bioactive compounds, with emphasis on the

flavonoids, such as naringin, hesperidin, neohesperidin, neoeriocitrin,

nobiletin, tangeretin, and naringenin as compiled in this review. In

fact, these compounds are believed to play a very significant role in

reducing the levels of total cholesterol, triglycerides, and LDL

(Mulvihill and Huff, 2012; Assini et al., 2013; Kou et al., 2017;

Zeka et al., 2017). Several studies have shown that naringin reduces

the HMGR activity more potently than does vitamin E (Choi et al.,

2001; Lee et al., 2001), as well as decreasing the action of ACAT (Kim

et al., 2006), which contributed to hypocholesterolemic action and

higher excretion of fecal sterols (Jeon et al., 2004). Similarly,

hesperidin reduces plasma cholesterol in hypercholesterolemic rats

by decreasing ACAT and HMGR (Lee et al., 1999; Lee et al., 2012)

besides changing the expressions of genes encoding PPARs and the

LDL receptor (Akiyama et al., 2009). A recent study demonstrated

that neohesperidin is also able to regulate the lipid metabolism in vivo

and in vitro via FGF21 and AMPK/SIRT1/PGC-1α signaling axis

(Wu et al., 2017). Furthermore, the non-glycoside Citrus flavonoid,

naringenin, stimulates the hepatic fatty acid oxidation via PPARγ and

prevents lipogenesis in both the liver and the muscle, reducing the

serum lipid levels (Mulvihill et al., 2009).

FIGURE 6 | Biochemical and tissue changes caused by diets high in fat and calories (black lines) and mechanisms of action of Citrus products upon metabolic

disorders associated with hyperlipidemia (blue lines indicate activation and red lines indicate inhibition).

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia

In this review, we also observed that the Citrus products act by

reducing the atherogenic index or tissue manifestations associated

with atherosclerosis (Vinson et al., 1998; Bok et al., 1999; Zulkhairi

et al., 2010). In fact, the polyphenolic compounds and flavonoids

found in the Citrus species have antioxidant (Vinson et al., 1998;

Gorinstein et al., 2007; Zulkhairi et al., 2010; Craft et al., 2012) and

anti-inflammatory properties, in addition to their ability to decrease

LDL levels, inhibiting the formation of atherosclerotic plaques (Tripoli

et al., 2007; Assini et al., 2013; Onakpoya et al., 2017). Naringin, for

example, reduces plaque progression once it decreases non-high-

density lipoprotein cholesterol concentrations and biomarkers of

endothelial dysfunction and inhibits the expression of ICAM-1 in

endothelial cells, preventing immune cell adhesion and infiltration in

the vascular wall (Choe et al., 2001; Chanet et al., 2012).

Confirming the results of the systematic review, the meta-analysis

of preclinical studies indicated that Citrus products reduce the total

cholesterol, triglycerides, and LDL levels by −41.76, −44.28, and

−27.45 mg/dL, respectively, while increasing the HDL levels by

4.25 mg/dL. Similar results were observed in the clinical studies, in

which the Citrus species induce a reduction in the total cholesterol,

triglycerides, and LDL levels by −42.03, −62.41, and −37.76 mg/dL,

respectively, whereas the HDL levels increased by an average of

5.85 mg/dL.

In the meta-analysis published by Onakpoyaa et al. (2015)

(Onakpoya et al., 2017), performed with two clinical trials

about the effect of grapefruits on the lipid profile, significant

effects were observed only for the increase in HDL, without TC

and LDL changes. More recently, a meta-analysis published by

Kou et al. (2017) showed that the sizes of effect measures for

LDL and total cholesterol presented significant results in the

group

of

patients

treated

with

Citrus

juice,

without

considerable changes in HDL and TG levels. The divergence

between the results presented in our meta-analysis compared

to those previously published is justified by the broader scope

of our question, as well as the inclusion of more recent studies,

which have confirmed the contribution of Citrus-based

products in the control of blood lipids.

Through the analysis of the risk of bias, it can be observed

that the preclinical studies have a satisfactory average score,

with some limitations in the methodological description of the

studies

and

the

results.

Similarly,

clinical

studies

had

limitations

in

reporting

or

methodology

in

terms

of

blinding, allocation, randomization, and reporting of results.

The use of tools to assess the risk of bias in the studies included

in the systematic reviews has been widely well supported by

groups such as SYRCLE (Hooijmans et al., 2014), ARRIVE

(Kilkenny et al., 2010), and Cochrane (Cochrane Training,

2019), since the credibility of the results and the strength of the

evidence depend on the methodological criteria of the studies

(Busch et al., 2020).

Thus, although the results obtained are favorable to the treatment

with Citrus extracts, the methodological limitations and high

heterogeneity of the studies included in the meta-analysis weaken

the evidence about the real benefits of this intervention. In addition,

the studies do not provide information on effective dose,

bioavailability, efficacy, and safety. These parameters are required

to propel the use of these promising therapeutic agents into the

clinical area. For this reason, further studies are needed to strengthen

the evidence of the effects of Citrus on dyslipidemia.

This systematic review presents as limitations the low evidence

found due to the high variability of the studies and variation of the

methodological protocols of the articles. Among them, we can

mention the differences in the induction of dyslipidemia, routes

of administration, and types of extracts, besides the absence of

baseline serum levels of lipids for comparison after the induction

and inconclusive report. Finally, as in our review, of the 25 studies

included in the meta-analysis, only 3 presented results in humans; we

chose not to use the GRADE system. For this reason, we believe that

further clinical studies are needed to provide sufficient scientific

support

to measure

the

effectiveness of

Citrus

effects

on

dyslipidemia.

CONCLUSION

From the compilations of the studies, one can suggest that the

Citrus extract has a potential effect in dyslipidemia control, both

in the preclinical studies and clinical trials. These effects can be

associated with the presence of bioactive compounds, as

flavonoids, which act synergistically through several pathways,

causing inhibition of lipogenesis and activating β-oxidation.

However, due to the high heterogeneity of the reposted

findings, further studies are needed to increase the strength of

clinical evidence of the action of Citrus extracts on the control of

dyslipidemia and increase the strength of that evidence.

DATA AVAILABILITY STATEMENT

The original contributions presented in the study are included in

the article/Supplementary Material; further inquiries can be

directed to the corresponding author.

AUTHOR CONTRIBUTIONS

Ideation and preparation of the review: BC and AG; search and

selection of studies: BC and LN; third evaluation for discrepancy

analysis: AG; qualitative data extraction: BC, LN, and JN; quantitative

data extraction: BC and VG; meta-analysis: BC, VG, and PZ; writing

and finalizing the review: BC, DT, and AG.

FUNDING

The authors acknowledge grants from the Foundation for Support

of Research and Technological Innovation of the State of Sergipe

(Fundação de Apoio à Pesquisa e Inovação Tecnológica do Estado de

Sergipe: FAPITEC/SE, EDITAL CAPES/FAPITEC/SE N^° 10/2016 –

PROMOB

1995/2017),

National

Council

for

Scientific

and

Technological

Development

(Conselho

Nacional

de

Desenvolvimento

Científico

e

Tecnológico:

CNPq/Brazil),

Coordination

for

the

Improvement

of

Personnel

Higher

Education (Coordenação de Aperfeiçoamento de Pessoal de Nível

Superior: CAPES/Brazil), and Federal University of Sergipe.

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia

ACKNOWLEDGMENTS

We are grateful for the support given by Patrícia K. Ziegelmann in

the elaboration of the meta-analysis and to teacher Abilio Borghi

for the assistance with English language review.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at:

https://www.frontiersin.org/articles/10.3389/fphar.2022.822678/

full#supplementary-material

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Conflict of Interest: The authors declare that the research was conducted in the

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Carvalho et al.

Citrus Extract as a Perspective for the Control of Dyslipidemia